Growth-stimulating material derived from porcine bone therefor and a manufacturing process

ABSTRACT

The invention relates to a therapeutic composition (especially for bone lesions with cavity formation and parodontitis) containing a growth-stimulating material and a solid carrier, to such a material and to a process for the preparation thereof.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a therapeutic composition for use in parodontaltreatment.

According to the findings of the WHO, inflammatory diseases of theteeth-holding system (parodontitis, often also called "parodontosis")are amongst the most widely distributed chronic diseases The long-termpreservation of teeth is endangered by the disintegration of the jawbone occurring in these diseases (especially in the form of deep bonecavities).

Although the previously propagandized rather radical methods involvingrecontouring of the bone and the soft tissue provided a result thatcould readily be checked, at the same time parodontal regeneration wasnot attempted and occasionally even the loss of healthy parodontaltissue was caused;

2. Brief Description of the Prior Art

The aim of modern parodontal treatment is the regeneration of parodontaltissue by cleaning the roots of teeth to remove microbial plaque(concrements), and to remove from the bone cavities tissue modified byinflammation. Numerous methods of treatment of parodontal lesions havebeen developed for this; cf., inter alia, Prichard 1975, Rosling et al.1976, Polson et al. 1978, Ramfjord & Ash 1979 and Rateitschak et al.1984. A certain parodontal regeneration should occur either by merelycleaning the osseous lesions, or by implanting various materials intothe bone cavities; The implantation material preferably used today ishydroxyapatite which, according to clinical tests, appears to bringabout a greater repair of the lesion than simple root and lesioncleaning;

In all of the cases mentioned, however, there is one fundamentalproblem. Daughter cells growing out from the oral epithelium form alongthe surface of the root a new epithelium, which in most cases covers thewhole of the treated surface of the root and later may even lie at theapex of the regenerated bone. This epithelium may promote a newinflammation in the depths of the treated bone lesion. The basicproblem, however, is that the deep growth of the epithelium proceedsmore quickly than does the osseous and desmodontal regeneration.

In order to prevent this deep growth of the epithelium, according tomore recent research the bone lesion was covered, for example, withmillipore filter before the mucoperiosteal flap was put back inposition. This is intended to render possible parodontal regenerationwithout interference from deep growth of the epithelium. This processappears very complicated, however, and the prognosis is uncertain as aresult of a high quota of possible errors.

SUMMARY OF THE INVENTION

One aim of the invention is to provide a therapeutic composition withwhich the regeneration time is reduced and the risk of deep growth ofthe epithelium is prevented.

This object is achieved in accordance with the invention by atherapeutic composition having a growth-stimulating material and a solidcarrier.

The person skilled in the art is familiar with growth-stimulatingmaterials; attention is drawn to the prior art, which is referred to inthe following in connection with a growth-stimulating material accordingto the invention that is especially suitable.

Growth-stimulating materials are available in liquid or paste form,their growth-stimulating action being attributable to proteins. Thepurpose of the carrier provided in accordance with the invention is tobind the growth-stimulating material so as to prevent it from beingwashed away from the site of application.

Examples of carriers that can be used in accordance with the inventionare partially to fully absorbable carriers, especially hydroxyapatite(HAP), tricalcium phosphate, collagen and plastics. As a result of theabsorbability of the carrier, the therapeutic composition according tothe invention can disappear at the site of application, whilst the boneis built up again by the action of the growth-stimulating material.

Growth-promoting activities of bone material have been described byvarious research groups; see Simpson E. (1984) TIBS 9, pp. 527-530 andCanalis E. (1983) Endocrine Reviews 4, pp. 62-77.

The research group Baylink et al. isolated from human material agrowth-promoting substance having a high molecular weight of 83 kD(hSGF=human Skeletal Growth Factor), which promoted the growth ofosteoblast-analogous cells from hen embryos with a high content ofalkalne phosphatase; see Farley et al. (1982) Biochemistry 21, pp.3502-3507 and Farley et al. (1982) Biochemistry 21, pp. 3508-3517. Atthe same time, the dry weight of embryonic hen bones in organ culturewas increased in comparison with untreated control cultures by theaddition of hSGF. In a later publication, the target cells, which reactpositively to this growth-promoting substance or human factor, werecomprehensively described. Apart from the osteoblast-analogous cellsalready mentioned, skin and cartilage cells of embryonic hens, humanbone cells and skin cells, human osteosarcoma cells and Balb/3T3 cellswere also described as being positively stimulable; see Mohan et al(1984) Calcif. Tissue Int. 36, pp. 139-145.

Furthermore, there has also been described; see Jennings et al. (1985)Proc. Fed. Am. Soc. Exp. Biol. 44, p. 1099, No. 4025 the isolation frombone material of cows of a growth-promoting substance (bSGF=bovineSkeletal Growth Factor) having a molecular weight, after reduction withβ-mercaptoethanol, in the range of from 11 to 12 kD. The Urist et al.research group isolated from bovine bone a protein having a molecularweight of 18.5±0.5 kD that induces bone formation in living animals.This growth-promoting substance (bBMP=bovine Bone Morphogenetic Protein)was administered into muscle material of living mice; see Urist et al.(1984) PNAS 81, pp. 371-375 where it induced bone formation; also, bonelesions of cranial bone in dogs were treated by the administration ofbBMP.

A similar growth-promoting substance (hBMP=human Bone MorphogeneticProtein) having a molecular weight of 17.5 kD was isolated from humanbone material by the same research group; see Klausner A (1985)Biotechnology 3, pp, 507-511.

The Seyedin et al. research group; see Seyedin et al. (1985) PNAS 82,pp. 2267-2271 isolated two proteins from bovine bone which induced invitro the formation of cartilage cells from cells of mesodermal origin(CIF-A and CIF-B=Cartilage Inducing Factor A and B). The molecularweight of CIF-A and CIF-B was quoted in each case as 26 kD undernon-reducing conditions.

The isolation from bone material of rats of a growth-promoting substance(rSGF=rat Skeletal Growth Factor) having a molecular weight of from 6 to17.5 kD has also been described. The research group of Canalis et al.isolated from an in vitro culture medium of foetal rat cranial bone twoproteins (BDGF-1 and BDGF-2) which stimulated the growth of cells ofmesodermal origin in organ culture; see Canalis et al. (1980) Science210, pp. 1021-1023. The molecular weights of these proteins from theculture medium were quoted as 10 kD for BDGF-1 and from 25 to 30 kD forBDGF-2. Only BDGF-2 possessed a mitogenic action.

Growth stimulation of cells

The factor from human tissue isolated by Baylink et al. (hSGF) effectsespecially a stimulation in the growth of calvaria cells that have ahigh content of alkaline phosphatase Farley et al., supra.

The activity of bBMP was not tested in monolayer cultures; Urist et al.,supra. and Klausner supra. CIF-A and CIF-B cause an increase in thegrowth of chondrocytes, but this increase in growth was not tested inmonolayer cultures Seyedin et al., supra.

The target cells for BDGF-2 were not described in detail; Mohan et al.,supra and Canalis et al., supra.

Growth stimulation of bone

hSGF causes an increase in the dry weight of embryonic tubular bone inorgan culture; see Farley et al., supra, but which types of cell atwhich parts of these organ cultures have been promoted in their growthwas not indicated for hGSF.

No results that relate to in vitro organ cultures of embryonic tubularbone have been published for bBMP, CIF-A, CIF-B and BDGF-2.

Temperature estability of growth stimulation

hSGF is described as stable in a temperature range of from 55° to75°C.³, the investigations concerning this property having been carriedout on osteoblast-analogous cells see Farley et al., supra.

There is no data available on bBMP concerning this property. CIF-A andCIF-B, on the other hand, are stable for 3 minutes at 100° C.

Heat stability is mentioned for BDGF-2, Mohan et al., supra but novalues are given.

pH stability

hSGF is stable in a pH range of from 2.5 to 10, but no target cells onwhich these properties were tested were given see Farley et al., supra.

There are no published tests on the acid stability of bBMP, CIF-A andCIF-B.

BDGF-2 is acid-stable, see Mohan et al., supra, but no data on the pHrange or the target cells were published.

Molecular weight range

hSGF appears to exist in two forms. A high molecular weight form of hSGFexists in 25 mM phosphate buffer (pH 7.2) see Farley et al., supra. Onthe other hand, after treatment with 4M guanidinium hydrochloride,reversed phase high pressure liquid chromatography and reduction, bSGFis indicated as having a molecular weight of from 11 to 12 kD Jenningset al. supra. There are no details for hSGF and bSGF regarding the useof gel filtration columns with 4M guanidinium hydrochloride as eluant.

CIF-A and CIF-B have an apparent molecular weight of 26 kD eachaccording to gel electrophoresis see Seyedin et al., supra.

There are similarly no details on the molecular weight of BDGF-2 usinggel filtration columns with 4M guanidinium hydrochloride as eluant.

Behaviour on copper (II) sulphate-chelate columns

There are no details on the behaviour of hSGF, bSGF, bBMP, CIF-A, CIF-Band BDGF-2 on copper(II) sulphate-chelate columns.

Behaviour on reversed phase columns

bSGF can be bound to reversed phase columns (Reversed Phase HPLC) butcan be eluted again with an acetonitrile gradient; see Jennings et al.,supra, there are no details on the range of concentration. bBMP, CIF-Aand CIF-B can also be bound to a reversed phase column and eluted withan acetonitrile gradient; see Urist el al., supra. and Seyedin et al.,supra.

There are no details on the binding capacity of BDGF-2 to reversed phasecolumns.

Extraction

Various extraction processes have been described for the mentioned Stateof the Art growth-promoting substances.

Ends of human femorae have been used as the bone material for isolatinghSGF; Farley et al., supra. Extraction with demineralisation was carriedout using EDTA (10%). The manner in which the bone material wascomminuted was not published.

For bSGF it is mentioned that an EDTA extract was obtained from bovinebone, but the bone was not specifically defined; Jennings et al., supra.There are no details concerning the manner in which the bone materialwas comminuted. bBMP was obtained from tubular bone. The bones, cooledin liquid nitrogen, were ground to meal in a Whiley mill, freed from fatand washed. The meal was subsequently demineralised with 0.6M HCl; seeUrist et al., supra. CIF-A and CIF-B were obtained from metatarsalbovine bone. The bones were pulverised in a mill cooled by liquidnitrogen In this case demineralisation was with 0.5M HCl. Extraction wascarried out with 4M guanidinium hydrochloride/10 mM EDTA; see Seyedin etal., supra. BDGF-2 does not have to be extracted since this factor isreleased by embryonic tissue into the surrounding culture medium; seeCanalis et al., supra.

rSGF was extracted from not specifically defined tubular bones of ratsusing a mixture of 4M guanidinium hydrochloride/EDTA (10%); see Sibongaet al. (1985) Proc. Fed. Am. Soc. Exp. Biol. 44, p. 1099, No. 4028.

Concentration

There is no data in the literature for hSGF relating to binding tohydroxyapatite.

There is no data in the literature concerning binding to hydroxyapatitefor CIF-A, CIF-B and BDGF-2. bBMP binds to hydroxyapatite but has notbeen tested either in organ culture or in monolayer cultures bSGF can beconcentrated by binding to hydroxyapatite. The process describedconsists of two steps:

1) binding to hydroxyapatite, elution with phosphate buffer;

2) binding to hydroxyapatite, elution with phosphate buffer in thepresence of 4M guanidinium hydrochloride.

For the total mitogenic activity according to these two steps, aconcentration factor of approximately 14 for a total yield of mitogenicactivity is indicated, but this cannot be clearly calculated from thedata in the literature; see Jennings et al., supra.

rSGF binds to hydroxyapatite, but no exact conditions have been given;see Sibonga et al., supra.

Growth-promoting factors of bone material from pigs are not so farknown.

Another object of the invention is to provide a growth-stimulatingmaterial that is particularly suitable for the therapeutic compositionof the invention.

This object is achieved by a growth-stimulating material consisting ofor comprising one or more growth-promoting (mitogenic) factors that canbe obtained by the invention the steps of claim 8 and optionally, inaddition, the steps of claim 9 and optionally claim 10, and that ischaracterised by the features of the characterising clause of claim 2.

The invention also relates to a process for obtaining growth-stimulatingmaterial.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION Microtitreplate test system

The growth-stimulating material according to the invention is tested inmicrotitre plates in an optimised test system automated as regards thecell harvest. This test system provides the following steps:

(a) The incorporation of ³ H-labelled thymidine into the total DNA ofosteoblast precursor cells is carried out by plating out theseosteoblast precursor cells onto microtitre plates, without change ofmedia, in a concentration of foetal calf's serum (FCS) that is less than0.5% (calculated by volume).

(b) The cell density of osteoblast precursor cells, which respond to theaddition of the growth-stimulating material of the invention having theactions and properties the invention by an increase in the incorporationof ³ H-thymidine into the total DNA, is in the range of from 2,500 to15,000 cells per microtitre well (6 mm diameter) at the time of platingout.

(c) The maximum response (incorporation of ³ H-thymidine into the totalDNA after the addition of the growth-stimulating material of theinvention having the actions and properties of the invention is recordedat a cell density of 5,000 osteoblast precursor cells per microtitrewell (6 mm diameter) at the time of plating out and after growth ofthese osteoblast precursor cells for 4 days and with an incubation offrom 16 to 22 hours.

(d) The incorporation of ³ H-thymidine into the total DNA after theaddition of the growth-stimulating material according to the inventionhaving the actions and properties according to the invention is, in thecase of chondrocytes in microtitre plates (6 mm diameter per microtitrewell) at an FCS concentration of less than 0.5% (calculated by volume),at a cell concentration of from 1,250 to 10,000 chondrocytes permicrotitre well at the time of plating out, increased on the first,second and third day after plating out.

(e) The incorporation of ³ H-thymidine into the total DNA of adherentcell cultures in microtitre wells is achieved by the following method.The adherent cell cultures are lysed in the microtitre wells afterremoval of the radioactive medium and after the washing operations.Subsequently, the total DNA is precipitated in the microtitre wells byacid. The precipitated total DNA and the radioactivity (³ H-thymidine)contained in it are transferred simultaneously from several mircotitrewells onto filter paper using an automatic cell-harvesting device Therate of incorporation is then determined.

In the following the invention is explained in detail by way ofExamples.

EXAMPLE 1

50 kg of bone material from pigs was freed from skin, flesh and fat at+4° C., cooled in liquid N₂ and stored at -20° C. The bone material wascompressed with a compressing device while cooling with liquid N₂,resulting in a total yield of 10 kg of bone meal of a particle size offrom 0.5 to 2 mm.

1 kg of bone meal was dialysed for 2 weeks in dialysis tubes against 10%EDTA (weight/volume), and the dialysis solution was changed twice. Then,the supernatant solution in the dialysis tubes was homogenised with theinsoluble residue in a Star mix. The suspension was centrifuged twice,in each case the pellet was discarded, and the supernatant was heated at75° C. for 15 minutes. The precipitated protein was removed bycentrifugation and the supernatant was brought to a pH of 3.0 with 1NHCl for 20 minutes. The precipitated protein was again removed bycentrifugation and the supernatant was brought to a pH of 7.2 with 1NNaOH. Dialysis was then carried out against 25 mM phosphate buffer, pH7.2. 780 ml of crude extract having a protein content of 218 μg/ml=170mg total protein were obtained. This crude extract was applied to ahydroxyapatite column having a diameter of 5 cm and a height of 30 cmand washed with 25 mM phosphate buffer (2 L) and with 380 ml of aphosphate gradient increasing from 25 mM to 100 mM. Elution was thencarried out with 4M guanidinium HCl. The active fractions from theelution with 4M guanidinium.HCl were pooled, dialysed against 4Mguanidinium.HCl and applied to a gel filtration column, with 4Mguanidinium.HCl as eluant, which column had previously been calibratedwith standard proteins. The active fractions in a molecular weight rangeof 24±8 kD were eluted from this gel fitration column. The activefractions of the hydroxyapatite column and the gel filtration columnwere dialysed against 25 mM phosphate buffer. The dose-dependentincrease in the incorporation of ³ H-thymidine into DNA of osteoblastprecursor cells was determined for the products of the individualpurification steps. One unit was defined as the protein concentration atwhich the semi-maximum stimulation of these cells in the incorporationof ³ H-thymidine occurs. The following values were obtained:

    ______________________________________                                               specific           total                                                      activity  total    protein   concen-                                          (units/μg)                                                                           units    (μg)   tration                                   ______________________________________                                        crude extract                                                                            13        2,210,000                                                                              170,000  1                                      hydroxy-   500       1,547,000                                                                               3,094   38                                     apatite                                                                       gel filtra-                                                                            3,300         330,000                                                                                 100  254                                     tion                                                                          ______________________________________                                    

EXAMPLES 2 AND 3 AND COMPARISON EXAMPLES 1 AND 2

In an animal experiment carried out on a narcoticised dog, the bone inthe lower jaw region was exposed by preparation of a mucoperiostealflap. Then, boreholes (diameter 2.8 mm, depth 5 mm) were made with astandard internally cooled implant drill. These holes were filled with apreparation of hydroxyapatite, with growth-stimulating materialaccording to Example 1 or with sodium chloride, in the amountsindicated:

    ______________________________________                                               Preparation                                                                   HAP      Growth-stimu-                                                                             NaCl                                                     (parts   lating material                                                                           (parts                                                   by wt.)  (parts by vol.)                                                                           by wt.)                                           ______________________________________                                        Example                                                                       2        250 × 10.sup.3                                                                     50          0                                             3        250 × 10.sup.3                                                                     50          0                                             Comparison                                                                    Example                                                                       1        250 × 10.sup.3                                                                      0          50                                            2        0           0          0                                                                             Hole remained                                                                 exposed                                       ______________________________________                                    

After eight weeks the hole in each of Examples 2 and 3 and ComparisonExample 2 was completely sealed, whilst in Comparison Example 1, in theupper region only osseous regeneration with the exception of the centralthird had taken place. It can be concluded from this that thegrowth-stimulating material in Examples 2 and 3 caused an improvement inthe new bone formation as compared with Comparison Example 1.

EXAMPLES 4 AND 5 AND COMPARISON EXAMPLES 3 AND 4

Examples 2 and 3 and Comparison Examples 1 and 2 were repeated and theevaluation was carried out after 3 weeks.

In Example 4 the hole exhibited in the upper region and basally, as faras half way, a spongy lamellar new bone formation (with integration ofthe peripheral preparation particles).

In Example 5 there was a lamellar new bone formation with integration ofthe preparation particles in the outer third to half of the upper regionof the hole, and basally the entire hole was almost completely filled bytrabeculae with integration of the preparation particles.

In Comparison Example 3, it was possible to observe new bone formationin the upper region of the hole in the outer third of half of thecircumference without incorporation of the preparation particles;basally there was new bone formation in the outer half with partialincorporation of the preparation particles which were fibrouslycentrally separated.

In Comparison Example 4, newly formed trabeculae filled the hole of onespecimen almost completely and the hole of another specimen up to thecentral third.

We claim:
 1. A composition which comprises:I an active ingredientprepared by(a) cooling in liquid nitrogen fat-free bone material ofmetacarpalia from pigs; (b) comminuting the bone material at thetemperature of liquid nitrogen, to bone meal of a particle size in therange from 0.5 to 2 mm; (c) demineralizing the bone particles in anaqueous solution; and separating from the resulting solution, the activeingredient having the following features:(a) the active ingredientcauses a dose-dependent growth stimulation of fibroblasts, osteoblastprecursor cells and chondrocytes and embryonic tissue of chickens inmonolayer cell cultures; (b) the active ingredient causes adose-dependent growth of embryonic tubular bones of chickens in organculture in vitro, due to a proliferation of chondrocytes in the growthzone; (c) the active ingredient does not cause any increasedincorporation of ³ H-thymidine into DNA of calvaria cells in themicrotitre plate test system; (d) the feature (a) or (b) above areretained for at least 15 minutes at a temperature of from 55° to 75° C.;(e) the features (a) or (b) above are retained for at least 20 minutesat a pH of approximately 3 and of approximately 10; (f) the activeingredient can be eluted on a gel filtration column under high saltconditions in a molecular weight range of 24±8 kD; (g) the activeingredient runs through a copper sulphate-chelate column without beingadsorbed, it being possible for the specific activity to be concentratedby a factor of from 5 to 20; (h) the active ingredient can be bound to areversed phase column in high pressure liquid chromatography and elutedwith an acetonitrile/water mixture having an acetonitrile content in therange of from 10 to 45% calculated by volume; (i) 100 to 200 pg of theactive ingredient forms more than 5 mm³ bone mass growth; and (ii) theactive ingredient can be dialysed against 25 mM phosphate buffer; and IIa solid carrier to bind the active ingredient to the site ofapplication.
 2. A composition according to claim 1 wherein the carrieris a partially to fully absorbable carrier.
 3. A composition accordingto claim 1 having a ratio of active ingredient material:solid carrier of1:1000 to 1:200,000 (volume:weight).
 4. A process for obtaining theactive ingredient according to claim 1, which comprises;cooling inliquid nitrogen fat-free bone material of metacarpalia from pigs;comminuting the bone material to bone meal of a particle size in therange of from 0.5 to 2 mm at the temperature of liquid nitrogen;demineralizing the bone meal particles with an aqueous solution;dialysing the resulting aqueous suspension to extract the activeingredient; acidifying the supernatent to an acid pH; heating theacidified supernatent to a temperature sufficient to precipitateprotein; and centrifuging off insoluble components.
 5. The processaccording to claim 4, wherein, the aqueous supernatant obtained aftercentrifugation isapplied to a chromatography column of hydroxyapatiteand eluted with guanidinium hydrochloride.
 6. The composition of claim 1wherein the solid carrier is selected from the group consisting ofhydroxyapatite, tricalcium phosphate, and collagen.
 7. The compositionof claim 1 wherein separation of the active ingredient comprisesdialysis, acidification of the dialysis supernatent, heating of thesupernatent and centrifugation to remove insolubles.
 8. The compositionof claim 7 wherein the separated active ingredient is applied to ahydroxyapatite column and eluted with guanidinium hydrochloride.
 9. Thecomposition of claim 8 wherein the eluate is applied to a gel filtrationcolumn and eluted under high salt conditions in a molecular weight rangeof approximately 24±8 kD.
 10. A composition useful as a mitogenicfactor, which is prepared by;(a) cooling in liquid nitrogen fat-freebone material of metacrapalia from pigs; (b) comminuting the bonematerial at the temperature of liquid nitrogen, to bone meal of aparticle size in the range from 0.5 to 2 mm; (c) demineralizing the boneparticles in an aqueous solution; (d) dialyzing the aqueous solutionagainst a 10 percent (weight/volume) solution ofN,N,N',N',-ethylenediaminetetraacetic acid; (e) heating the supernatentat a temperature of 75° C. for 15 minutes to precipitate protein; and(f) acidifying the solution to a pH of 3.0; and (g) centrifuging toremove precipitated protein; said composition having the followingfeatures:(a) a dose-dependent growth stimulation of fibroblasts,osteoblast precursor cells and chondrocytes and embryonic tissue ofchickens in monolayer cell cultures; (b) a dose-dependent growth ofembryonic tubular bones of chickens in organ culture in vitro, due to aproliferation of chondrocytes in the growth zone; (c) do not cause anyincreased incorporation of ³ H-thymidine into DNA of calvaria cells inthe microtitre plate test system; (d) the feature (a) and (b) above areretained for at least 15 minutes at a temperature of from 55° to 75° C.;(e) the features (a) or (b) above are retained for at least 20 minutesas a pH of approximately 3 and of approximately 10; (f) the compositioncan be eluted on a gel filtration column under high salt conditions in amolecular weight range of 24±8 kD; (g) the composition run through acopper (11) sulphate-chelate column without being adsorbed, it beingpossible for the specific activity to be concentrated by a factor offrom 5 to 20; (h) the composition can be bound to a reversed phasecolumn in high pressure liquid chromatography and eluted with anacetonitrile/water mixture having an acetonitrile content in the rangeof from 10 to 45%; (i) 100 to 200 μg of the active ingredient forms morethan 5 mm³ bone mass growth; and (j) the active ingredient can bedialysed against 25 mM phosphate buffer; and II a solid carrier to bindthe active ingredient to the site of application.
 11. The composition ofclaim 10 wherein after centrifuging, the supernatent is applied to achromatography column of hydroxyapatite and eluted with guanidiniumhydrochloride and the eluate is applied to a gel filtration column andeluted with 4M guanidinium hydrochloride in a molecular weight range ofapproximately 24±8 kD.